We will develop clinical diagnostics tests for two Category B agents (Burkholderia pseudomallei and B. mallei) based upon real-time PCR low density microarrays and MLVA in order to provide rapid and accurate information to physicians. The etiological agent of melioidosis is rarely seen in US clinics but causes substantial morbidity and mortality in many areas of the world, including Australia and Southeast Asia. Glanders is a zoonotic disease and likewise is rarely seen in the US. While these diseases are difficult to diagnose, early and accurate detection and identification of these diseases can have a great impact on therapeutic response and disease outcomes. The development of novel diagnostic assays (real time-PCR) for these pathogens is crucial, as the current assays used in clinical settings lack the speed, sensitivity and specificity needed to address these highly fatal illnesses. The phylogenetic assay (MLVA) will provide specific information regarding the chronic status of cases, as well as the potential genetic linkage between cases. Development of these assays will translate into both clinical and public health settings, in either single pathogen assay kits or in a multiple-pathogen low density array format. These assays will provide identification, quantitative, and clinically-important qualitative data (e.g., antibiotic resistance, virulence and chronic infection markers). Our research strategy involves a multi-faceted translational collaboration, designed to optimize the move from research discovery to clinical application. The collaborators in this activity include a non-profit research institute (TGen), a university (NAU), a clinical partner (Menzies/Royal Darwin Hospital -Australia), and an industrial/diagnostics manufacturing partner (Applied Biosystems). This translational strategy has proven successful in other activities, including a current project involving three of the above partners (TGen, NAU and AB). Additionally, TGen and NAU have successfully collaborated with Menzies on other Burkholderia-re\a\ed activities, paving the way for a seamless consortium on this project. The proposed activity will move in a logical sequence: A) Signature Identification (Specific Aim #1) B) Conversion of Signatures to Assays (Specific Aim #2); C) Assay validation (Specific Aim #3); D) Assay Manufacturing (Specific Aim #4) E) Clinical Sample Preparation (Specific Aim #5) F) Clinical Evaluation of Assays (Specific Aim #6) G) High Resolution Genetic Analysis of Isolates (Specific Aim #7) H) Research Information Management (Specific Aim #8) The members of our collaborative team are highly experienced in each of these areas, as well as with these pathogens and their diseases. In short, the proposed research and development will result in novel laboratory tests, using state of the art genetic technology, for identifying and describing infections with the pathogens that cause melioidosis and glanders, Burkholderia pseudomallei and B. mallei respectively. The importance of the development of these new assays (tests) will be felt by the medical and public health community, as they will allow for faster diagnoses, more information about cases, better pathways to treatments, and improved epidemiological information for disease control activities.